PROJECT SUMMARY Human immunodeficiency virus (HIV) infection is characterized by depletion in CD4+ T cells and persistent immune activation as a result of epithelial barrier disruption and systemic translocation of microbial products. Immune targets of HIV such as CD4+ T cells, macrophages, and dendritic cells have been the main research focus for many years, but modifications in these cell types have not explained HIV-induced epithelial damage. For this reason, better understanding of epithelial cell metabolism and interactions with gut microbiota has been an important aspect of intestinal barrier function in HIV. Studies have shown that treatment with probiotic microbiota can positively impact mucosal immune responses, support renewal of the gut epithelial barrier, and suppress inflammation. We utilized injections of probiotic L. plantarum in a ligated ileal loop model in chronic SIV-infected rhesus macaques to investigate mechanisms of epithelial damage and repair. Through a metabolomics approach, I found that epithelial cell damage may be linked to malonate accumulation, activating transcription factor NF-kB and downstream epithelial damage. Treatment with L. plantarum reduced levels of malonate in the gut and repaired epithelial integrity. We want to investigate the role of malonate metabolism in the gut to answer mechanistic questions regarding the role of microbiota in modulating gut barriers and the immune system in HIV and other inflammatory diseases. First, we will test the source of malonate overproduction in HIV infection using cell culture models and proteomic analysis. Then, we will assess the molecular mechanisms of NF-kB activation by malonate accumulation and determine if inhibiting this pathway genetically could prevent phenotypic changes in epithelial integrity. Finally, we will evaluate the ability of L. plantarum to reduce epithelial damage through malonate reduction. The outcomes from this study will address the immunometabolomic connection between gut microbiota and host cell function in HIV infection. Identifying regulatory elements of chronic gut inflammation and damage will be an essential contribution to research in autoimmune or infectious diseasees targeting the gastrointestinal tract. Hypothesis 1: Epithelial barrier damage is induced by increased malonate production in response to HIV infection. Furthermore, we hypothesize that L. plantarum can restore epithelial barrier integrity by reducing malonate accumulation. Aim 1: To demonstrate that epithelial cells produce excess malonate in response to HIV infection Aim 2: To determine if malonate induces NF-kB activation through post-translational malonylation Aim 3: To investigate the ability of L. plantarum to repair epithelial damage through malonate reduction